Protein expression changes of HCN1 and HCN2 in hippocampal subregions of gerbils during the normal aging process

Document Type: Original Article


1 Department of Pharmacy, College of Pharmacy, Dankook University, Cheonan, Chungnam 31116, Republic of Korea

2 Department of Anatomy, College of Korean Medicine, Dongguk University, Gyeongju, Gyeongbuk 38066, Republic of Korea

3 Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea


Objective(s): Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play essential roles in various hippocampal functions, including regulation of long-term potentiation, synaptic plasticity, and hippocampal-dependent cognitive process. The objective of this study was to investigate age-related changes in HCN1 and HCN2 protein expressions in gerbil hippocampus at various ages.
Materials and Methods: In this study, the protein expressions of HCN1 and HCN2 were compared in the hippocampus at the ages of 1, 3, 12, and 24 months using Western blot analysis and immunohistochemistry.
Results: Immunoreactivity of both HCN1 and HCN2 was shown primarily in cells of the pyramidal cell layer in the hippocampus proper and in cells of the granule cell layer in the dentate gyrus. HCN1 and HCN2 protein expression levels and immunoreactivity were significantly increased at three months (3 M) of age compared with those at 1 M of age. After that, both HCN1 and HCN2 expression levels in the hippocampus were gradually decreased with age.
Conclusion: Our results show that the normal aging process affects the expression levels of HCN1 and HCN2 in hippocampal cells in gerbils. There are marked reductions in HCN1 and HCN2 expressions in the aged hippocampus compared to the young hippocampus. Such reductions might be related to aging in the hippocampus.


Main Subjects

1. Gallagher M, Bizon JL, Hoyt EC, Helm KA, Lund PK. Effects of aging on the hippocampal formation in a naturally occurring animal model of mild cognitive impairment. Exp Gerontol 2003; 38:71-77.
2. Geinisman Y, Detoledo-Morrell L, Morrell F, Heller RE. Hippocampal markers of age-related memory dysfunction: behavioral, electrophysiological and morphological perspectives. Prog Neurobiol 1995; 45:223-252.
3. Jacobson L, Zhang R, Elliffe D, Chen KF, Mathai S, McCarthy D, et al. Correlation of cellular changes and spatial memory during aging in rats. Exp Gerontol 2008; 43:929-938.
4. Enzinger C, Fazekas F, Matthews PM, Ropele S, Schmidt H, Smith S, et al. Risk factors for progression of brain atrophy in aging: six-year follow-up of normal subjects. Neurology 2005; 64:1704-1711.
5. Tombaugh GC, Rowe WB, Chow AR, Michael TH, Rose GM. Theta-frequency synaptic potentiation in CA1 in vitro distinguishes cognitively impaired from unimpaired aged Fischer 344 rats. J Neurosci 2002; 22:9932-9940.
6. Voglis G, Tavernarakis N. The role of synaptic ion channels in synaptic plasticity. EMBO Rep 2006; 7:1104-1110.
7. Dyhrfjeld-Johnsen J, Morgan RJ, Soltesz I. Double Trouble? Potential for hyperexcitability following both channelopathic up- and downregulation of I(h) in epilepsy. Front Neurosci 2009; 3:25-33.
8. Lewis AS, Chetkovich DM. HCN channels in behavior and neurological disease: too hyper or not active enough? Mol Cell Neurosci 2011; 46:357-367.
9. Lupica CR, Bell JA, Hoffman AF, Watson PL. Contribution of the hyperpolarization-activated current (I(h)) to membrane potential and GABA release in hippocampal interneurons. J Neurophysiol 2001; 86:261-268.
10. Magee JC. Dendritic Ih normalizes temporal summation in hippocampal CA1 neurons. Nat Neurosci 1999; 2:508-514.
11. Hussaini SA, Kempadoo KA, Thuault SJ, Siegelbaum SA, Kandel ER. Increased size and stability of CA1 and CA3 place fields in HCN1 knockout mice. Neuron 2011; 72:643-653.
12. Nolan MF, Malleret G, Dudman JT, Buhl DL, Santoro B, Gibbs E, et al. A behavioral role for dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal dendrites of CA1 pyramidal neurons. Cell 2004; 119:719-732.
13. Phillips AM, Kim T, Vargas E, Petrou S, Reid CA. Spike-and-wave discharge mediated reduction in hippocampal HCN1 channel function associates with learning deficits in a genetic mouse model of epilepsy. Neurobiol Dis 2014; 64:30-35.
14. Ludwig A, Zong X, Jeglitsch M, Hofmann F, Biel M. A family of hyperpolarization-activated mammalian cation channels. Nature 1998; 393:587-591.
15. Santoro B, Grant SG, Bartsch D, Kandel ER. Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels. Proc Natl Acad Sci U S A 1997; 94:14815-14820.
16. Monteggia LM, Eisch AJ, Tang MD, Kaczmarek LK, Nestler EJ. Cloning and localization of the hyperpolarization-activated cyclic nucleotide-gated channel family in rat brain. Brain Res Mol Brain Res 2000; 81:129-139.
17. Santoro B, Liu DT, Yao H, Bartsch D, Kandel ER, Siegelbaum SA, et al. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 1998; 93:717-729.
18. Santoro B, Tibbs GR. The HCN gene family: molecular basis of the hyperpolarization-activated pacemaker channels. Ann N Y Acad Sci 1999; 868:741-764.
19. Bender RA, Brewster A, Santoro B, Ludwig A, Hofmann F, Biel M, et al. Differential and age-dependent expression of hyperpolarization-activated, cyclic nucleotide-gated cation channel isoforms 1-4 suggests evolving roles in the developing rat hippocampus. Neuroscience 2001; 106:689-698.
20. Brewster AL, Chen Y, Bender RA, Yeh A, Shigemoto R, Baram TZ. Quantitative analysis and subcellular distribution of mRNA and protein expression of the hyperpolarization-activated cyclic nucleotide-gated channels throughout development in rat hippocampus. Cereb Cortex 2007; 17:702-712.
21. Santoro B, Chen S, Luthi A, Pavlidis P, Shumyatsky GP, Tibbs GR, et al. Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS. J Neurosci 2000; 20:5264-5275.
22. Seo H, Seol MJ, Lee K. Differential expression of hyperpolarization-activated cyclic nucleotide-gated channel subunits during hippocampal development in the mouse. Mol Brain 2015; 8:13-26.
23. Surges R, Brewster AL, Bender RA, Beck H, Feuerstein TJ, Baram TZ. Regulated expression of HCN channels and cAMP levels shape the properties of the h current in developing rat hippocampus. Eur J Neurosci 2006; 24:94-104.
24. Vasilyev DV, Barish ME. Postnatal development of the hyperpolarization-activated excitatory current Ih in mouse hippocampal pyramidal neurons. J Neurosci 2002; 22:8992-9004.
25. Choi HS, Ahn JH, Park JH, Won MH, Lee CH. Age-dependent changes in the protein expression levels of Redd1 and mTOR in the gerbil hippocampus during normal aging. Mol Med Rep 2016; 13:2409-2414.
26. Lee CH, Park JH, Choi JH, Yoo KY, Ryu PD, Won MH. Heat shock protein 90 and its cochaperone, p23, are markedly increased in the aged gerbil hippocampus. Exp Gerontol 2011; 46:768-772.
27. Hrapkiewicz KL, Stein S, Smiler KL. A new anesthetic agent for use in the gerbil. Lab Anim Sci 1989; 39:338-341.
28. Park JH, Kim YH, Ahn JH, Choi SY, Hong S, Kim SK, et al. Atomoxetine protects against NMDA receptor-mediated hippocampal neuronal death following transient global cerebral ischemia. Curr Neurovasc Res 2017; 14:158-168.
29. Huang CC, Hsu KS. Reexamination of the role of hyperpolarization-activated cation channels in short- and long-term plasticity at hippocampal mossy fiber synapses. Neuropharmacology 2003; 44:968-981.
30. Mellor J, Nicoll RA, Schmitz D. Mediation of hippocampal mossy fiber long-term potentiation by presynaptic Ih channels. Science 2002; 295:143-147.
31. Hongpaisan J, Xu C, Sen A, Nelson TJ, Alkon DL. PKC activation during training restores mushroom spine synapses and memory in the aged rat. Neurobiol Dis 2013; 55:44-62.
32. Murphy N, Cowley TR, Blau CW, Dempsey CN, Noonan J, Gowran A, et al. The fatty acid amide hydrolase inhibitor URB597 exerts anti-inflammatory effects in hippocampus of aged rats and restores an age-related deficit in long-term potentiation. J Neuroinflammation 2012; 9:79.
33. Yanai S, Ito H, Endo S. Long-term cilostazol administration prevents age-related decline of hippocampus-dependent memory in mice. Neuropharmacology 2018; 129:57-68.
34. Yang L, Zhang J, Zheng K, Shen H, Chen X. Long-term ginsenoside Rg1 supplementation improves age-related cognitive decline by promoting synaptic plasticity associated protein expression in C57BL/6J mice. J Gerontol A Biol Sci Med Sci 2014; 69:282-294.
35. Zamzow DR, Elias V, Shumaker M, Larson C, Magnusson KR. An increase in the association of GluN2B containing NMDA receptors with membrane scaffolding proteins was related to memory declines during aging. J Neurosci 2013; 33:12300-12305.
36. Small SA, Chawla MK, Buonocore M, Rapp PR, Barnes CA. Imaging correlates of brain function in monkeys and rats isolates a hippocampal subregion differentially vulnerable to aging. Proc Natl Acad Sci U S A 2004; 101:7181-7186.
37. Tsai SF, Ku NW, Wang TF, Yang YH, Shih YH, Wu SY, et al. Long-term moderate exercise rescues age-related decline in hippocampal neuronal complexity and memory. Gerontology 2018:1-11.
38. Li CJ, Lu Y, Zhou M, Zong XG, Li C, Xu XL, et al. Activation of GABAB receptors ameliorates cognitive impairment via restoring the balance of HCN1/HCN2 surface expression in the hippocampal CA1 area in rats with chronic cerebral hypoperfusion. Mol Neurobiol 2014; 50:704-720.
39. Luo P, Lu Y, Li C, Zhou M, Chen C, Lu Q, et al. Long-lasting spatial learning and memory impairments caused by chronic cerebral hypoperfusion associate with a dynamic change of HCN1/HCN2 expression in the hippocampal CA1 region. Neurobiol Learn Mem 2015; 123:72-83.